Myoglobin and haemoglobin: role of distal residues in reactions with haem ligands

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References 1 Maelicke, A. (1988) Trends" Biochem. Sci. 13, 199-202 2 Dohlman, H. G., Caron, M. G. and Lefkowitz, R. J. (1987) Biochemistry 26, 2657-2664 3 Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C. A., Neve, K. A. and Civelli, O. (1988) Nature 336,783-787 4 Civelli, O., Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Machida,

5 6 7 8

C. A. and Neve, K. A. (1989) in Molecular Biology of Neuroreceptors and Ion Channels (Maelicke, A., ed.), NATO ASI Series H, Springer Verlag (in press) Applebury, M. L. and Hargrave, P. A. (1987) Vision Res. 26, 1881-1895 Boyson, S. J., McGonigle, P. and Molinoff, P. B. (1986)J. Neurosci. 6, 3177-3188 Robertson, G. S. and Robertson, H. A. (1987) Trends Pharmacol. Sci. 8,295-299 Liibbert, H., Hoffman, B. J., Snutch, T. P.,

Myoglobin and haemoglobin: role of distal residues in reactions with haem ligands Despite the enormous amount of research done on the structure and function of haemoglobin, some of its most vital properties have remained ill understood. Free ferrous porphyrins are rapidly oxidized by oxygen, and their affinity for oxygen is several thousand times smaller than that for carbon monoxide. Globin keeps iron in the ferrous state which is necessary because only ferrous iron combines reversibly with molecular oxygen; globin also discriminates in favour of oxygen and against carbon monoxide. This is essential for life, since carbon monoxide is produced endogenously in the breakdown of porphyrin that follows the lysis of red cells. By a combination of directed mutagenesis and kinetic measurements Nagai, Siigar, Olson and co-workers have now got nearer to understanding how that discrimination works 1. The haem iron is linked to the globin by a bond to N~. of a histidine (FS) that acts as an electron donor in its reaction with oxygen. On its opposite or distal side, the haem faces a pocket that is lined with another histidine (E7) and a valine (El 1) (see Fig. 1). X-Ray analysis shows that the distal histidine blocks access to the haem pocket 2. Neither 02 nor CO can enter or leave unless the side chain of the distal histidine swings out of the way, which it can do only by elbowing the helix E, to which it is attached, away from the haem. Thus oxygen transport relies on the dynamics of the globin. In myoglobin and in the c~-subunits of haemoglobin, N~ of the distal histidine forms a hydrogen bond with the bound oxygen, but not with CO; in the 13-subunits that bond is either weak or absent 3'4 (Cheng, X. and Schoenborn, B. P. pcrs. commun.).

Using genetic engineering, Nagai, Sligar and co-workers have replaced the distal histidines in myoglobin and in the a- and [3-subunits of haemoglobin by glycines, which opened access to the haem pockets, and have measured the resulting changes in the rates of association and dissociation of O2, CO and with a more bulky ligand, methylisocyanide. Their results are best analysed in terms of transition state theory 5. If

van Dyke, T., Levine, A. J., Hartig, P. R., Lester, H. A. and Davidson, N. (1987) Proc. Natl Acad. Sci. USA 84, 4332-4336 9 Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M. and Nakanishi, S. (1987) Nature 329,836,838 ALFRED MAELICKE Max Pianck Institut for Ernfihrungsphysiologie, Rheinlanddamm 201, D-4600 Dortmund 1, FRG.

the transition state is product-like, any rise in affinity can be brought about mainly by a rise in the rate of association. If the transition state is reactantlike, any rise in affinity can be achieved mainly by a drop in the rate of dissociation. These rules generally hold, even though neither rate gives directly the rate of formation or dissociation of the transition state. The replacement of the distal histidine by glycine leaves the oxygen affinity and kinetic constants of the ]3-subunits unchanged within error; it diminishes the oxygen affinities of myoglobin 14-fold and that of the a-subunits eightfold, equivalent to stabilization of the bound oxygen by hydrogen bonds with the histidines by

is E7

Phe CD1

His

F8

Fig. 1. Arrangement of proximal and distal residues in myoglobin, showing the hydrogen bond between the distal histidine and the bound oxygen (from Ref. 3). The distal histidine has a pK, of about 5.5. Neutron diffraction has shown that in oxymyoglobin it is protonated only on N,:, in carbonmonoxymyoglobin only on N,~ which faces the surrounding water.

(~) Iq89,ElsevierSciencePublishersLkt. (UK} (137(~-5067i89/$(12.00

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T I B S 14 - February 1989

the equivalents of 1.4 and 1.0 kcal M-~ (5.9 and 4.2 kJ M-t), respectively. The reductions in affinity are brought about by 130- and 60-fold incre~tses in the dissociation (off) rates that more than compensate the 10-fold ir~creases in association (on) rates due to the opening of the haem pockets. The accelerations bring all the on-rates to values of 15(--+5) × 107 S - ! M ! which are close to Szabo's estimates of 50 x 107 s -I M l for a hypothetical globin in which that rate is limited only by diffusion into the haem pocket through a hole of 2.6 radius 5. The absence of any acceleration by the His---~Gly repl~Lcement in the [3-subunit implies that in native haemoglobin histidine E7I~ must be swinging in and out at leas1:10 9 times per second, while in myoglobin and in the c~-subunits that rate appears to be about a hundred times slower. All the o f f rates are several orders of magnitude slower than the on rates, which indicates that the rate-limiting step for the o f f rates is rupture of the F e - O bond rather than opening oF the haem pocket. All of these results apply only to the R structure of haemog [obin. Replacement of the dist~d histidine by glycine increases the affinity for CO fivefold in myoglobin, fourfold in the a-subunits and threefold in the ]3-subunits, due largely to increases in the on rates. In the native proteins, the on rates for CO are slower by ~Ln order of magnitude than those for O2, whence the acceleration is likely to be due to the removal of static steric hindrance by the distal histidines within the haem pocket rather than its fun,ztion as a gate. If we multiply the decrease in oxygen affinity by the increase in affinity for CO, we find that the distal histidine discriminates against CO by the equivalent of about 2 kcal M-~ (8.4 kJ M 1) in myoglobin and in the c~-subunits. The replacement of the distal histidine by glycine reduces the CO affinity of the [3-subunits to o he-third of its original value: this paradoxical result may be due to errors in measuring small changes in 9n and o f f rates, but even so the origin of the discrimination against CO in the 13-subunits remains a mystery. The switch in quaternary structure from R to T reduces the oxygen and CO affinities of haemoglobin by the equivalent of over 3 kcal (12.6 kJ) per mole haem. Like the reduction in oxygen affinity due to the His--~Gly replacement, it is brought about mainly by acceleration of the o f f rates. Hence all the evidence points to the transition

state with oxygen being mainly reactant-like. By contrast, the decrease in CO affinity in the R--*T transition is due to a drop in the on rates, consistent with the present evidence that transition state is mainly product-like. What role does the distal valine play in the discrimination between 02 and CO? Its replacement by alanine increases both the on and o f f rates of oxygen with the a-subunits sevenfold and leaves those with the 13-subunits unchanged. It increases the on rate of CO with the c~-subunits tenfold and leaves the o f f rates unchanged (Mathews, A. J., Rohlfs, R. J., Olson, J-S., Tame, J., Renaud, J-P. and Nagai, K., pers. commun.). Hence the distal valine in the c~-subunits discriminates against CO by the equivalent of 1.3 kcal M ~, apparently by steric hindrance, but is ineffective in the 13-subunits. However, this is only part of the story, because the results of Olson et al. are confined to the R structure L. In the T structure steric hindrance by the distal valine EII[3 plays a key role, which future experiments may quantify. Recent crystallographic studies indicate that the stereochemical basis for discrimination between oxygen and CO in haemoglobin may differ from that in myoglobin. In synthetic model compounds that offer no steric hindrance to the ligands, oxygen binds at an angle of 120° to the haem axis, while CO lies on the haem axis. The haem pockets of myoglobin and haemoglobin seemed to be tailored to accommodate the bent oxygen and force the CO off the haem axis, which might have accounted for their low CO affinity. This is true in myoglobin, where CO is seen in two orientations, with Fe-C-O inclined at either 120° or 140 ° to the haem axis 9. On the other hand, recent X-ray analysis of human carbonmonoxyhaemoglobins at 2.2-2.3 resolution have shown inclinations of less than 10 °, too little to account for the observed energy of discrimination. There are no significant displacements of the distal residues, but the porphyrins are ruffled (Emsley, P., Derewenda, S., Renaud, J-P., Dodson, G. and Perutz, M. F., pers. commun.). X-ray analysis of a synthetic 'hindered pocket' iron porphyrin that has a CO affinity lower than that of the unhindered 'picket fence' iron porphytin by the equivalent of 1.2 kcal (5 k J) per mole shows similar geometry. Fe-C-O is inclined to the haem axis by only 7.5 ° and the porphyrin is markedly ruffled l°. It looks as though in both the

'hindered pocket' porphyrin and in haemoglobin a major part of the strain energy is in the porphyrin. Computer simulations of the molecular dynamics of the exit of carbon monoxide from the interior of myoglobin, described by R. Elber and M. Karplus at a recent symposium*, suggest that there may be several alternative pathways in addition to that via the distal histidine v, even though the latter is the most direct. Experimental evidence in its support comes from the structure of phenylhydrazine myoglobin in which the side chain of the distal histidine has been turned out of the haem pocket by the bulk of the ligand 6 and from a recent crystal structure determination of ethylisocyanidemyoglobin (presented by G. N. Phillips, K. Johnson and J. S. Olson at the same meeting*) that shows the side chain of the distal histidine in two alternative positions, either in or out of the haem pocket, exactly as it would have to move to admit or release ligands. The dynamic movements of the haem pocket are attested by NMR studies 7 showing that phenylalanines CD1 and CD4, which wedge the haem into its pocket and are packed tightly between the haem and the distal helix E, flip over at rates faster than 104 s -~. They can do so only if the entire haem pocket breathes fast. Springer et al. 8 have studied the protection of the haem iron from oxidation by replacing the distal histidine in sperm whale myoglobin by ten different amino acid residues and shaking the deoxygenated myoglobin solutions in air in 75 m~ KPO4 + 25 mM E D T A pH 7.0 at 37°C. All replacements reduced the oxygen affinity and accelerated autooxidation. Phe, Met and Arg produced the smallest accelerations ( - 5 0 fold); Asp the largest (350-fold). How can these results be interpreted? Paradoxically, combination with oxygen protects the haem iron from oxidation, as can be shown by performing the same experiments at several atmospheres of pure oxygen. Apparently oxidation occurs in that fraction of molecules which are deoxygenated at any one moment. The larger that fraction is at the atmospheric oxygen pressure, the faster myoglobin autooxidizes. For example, replacement of the distal His by Phe reduced the oxygen affinity 170-fold, so *Symposium on oxygen binding heine proteins. Asilomar Conference Grounds, Pacific Grove, California, October 1988.

TIBS 14 - February 1989

44 that a larger fraction of myoglobin molecules will have remained deoxygenated at atmospheric oxygen pressure and therefore have become autooxidized. However, this can be only part of the explanation, because the replacement of His by Gly reduces the oxygen affinity merely ll-fold, yet accelerates autooxidation over a 100fold. Autooxidation is catalysed by protons, hence the 350-fold acceleration by Asp. Protons are reduced at the haem iron and in their turn reduce oxygen in the solvent to superoxide ion. I suggest that the distal histidine protects the ferrous haem iron by acting as a proton trap. The distal histidine has a pKa of about 5.5; at neutral pH it is protonated only at N6 which

faces the solvent. Any proton entering the haem pocket of deoxymyoglobin would be bound by N~, and simultaneously N6 would release its proton to the solvent. When the histidine side chain swings out of the haem pocket, the protons would interchange, restoring the previous state. No other amino acid side chain could function in this way. Evolution is a brilliant chemist. References 1 Olson, J. s. Mathews, A. J., Rohlfs, R. J., Springer, K . D . , Edeberg, K . D . , Sligar, S. G., Tame, J., Renaud, J. P. and Nagai, K. Nature 336,265-266 2 Perutz, M . F . and Mathews, F.S. (1966) J. Mol. Biol. 21,199 202 3 Phillips, S. E. V. and Schoenborn, B . P . (1981 ) Nature 292, 81-82 4 Shannan,B. (1983)J. Mol. Biol. 171,31-50

5 Szabo, A. (1978) Proc. Natl Acad. Sci. USA 75, 2108 6 Ringe, D., Petsko, G. E., Kerr, D. E. and de Montellano, P. R. O. (1984) Biochemistry 23, 2-4 7 Dalvit, C. and Wright, P. E. (1987) J. Mol. Biol. 194,313-327 8 Springer, B . A . , Egeberg, K . D . , Sligar, S. G., Rohlfs, R. J., Mathews, A. J. and Olson, J, S. J. Biol. Chem. (in press) 9 Kuriyan, J., Wilz, S., Karplus, M. and Petsko, G . A . (1986) J. Mol. Biol. 192, 133-154 10 Kim, K., Fettinger, J., Sessler, J. L., Cyr, M., Hugdahl, F., Collman, J. P. and Ibers, J. A. J. A m . Chem. Soc. (in press)

M. F. PERUTZ Laboratory of Molecular Biology, Hills Road, CambridgeCB2 2OH, UK.

Features Subcellular fractionation of tissue culture cells Kathryn E. Howell, Eileen Devaney and Jean Gruenberg Subcellular fractionation has two major steps, (1) the homogenization of" the cells and (2) the subsequent separation of the organelles. The homogenization step is discussed with reference to the problems encountered using tissue culture cells. Promising techniques for the isolation of specific compartments are illustrated using the isolation of the endosomal compartment as the example. Subcellular fractionation has been a basic research technique in Cell Biology for the last 30 years 1. The principles of the technique are clearly defined and best reviewed by Beaufay and Amar-Costesec 2. The analysis of subcellular fractions has produced a wealth of information and much of our present understanding of the structural-functional relationships of each of the organelles is based upon this accumulated knowledge 3. The methods used in classical subcellular fractionation were developed and perfected using tissues such as rat liver tn. Cultured cells have a number of advantages over the corresponding freshly extracted tissue for many experimental procedures and their use K. E. Howell and J, Gruenberg are at the European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, FRG. E. Devaney is now at the Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.

has greatly facilitated the development of new experimental approaches. However, tissue culture cells present a new set of problems in cell fractionation and much of the methodology developed for working with the rat liver or similar tissues is inappropriate. The major impediment to the successful fractionation of cultured cells is the difficulty of producing an ideal homogenate, that is, the release of all the organelles in suspension as individual elements, This may be explained 'by the differential organization of the cytoskeleton in cultured cells as compared with the corresponding tissue4'5 which results in the cytoplasm maintaining some degree of organization after homogenization. Organelles remain associated with the cytoskeletal elements surrounding the nucleus and become entrapped in 'clumps' of cytoplasm which readily sediment. As much as 50% of the components of the

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homogenate may be pelleted along with the nucleus during the initial centrifugation step 6-s. The cytoplasmic organization of different tissue culture cells varies enormously, so that homogenization conditions must be optimized for each cell line. Based on our experience with a number of different cell lines, including FAO, rat hepatocytes; BHK-21, baby hamster kidney; MDCK, Madin Darby canine kidney; and J774, mouse macrophage; we will discuss some of the problems encountered and suggest ways to produce appropriate fractions.

Homogenization After ideal homogenization particulate organelles, such as the nucleus, mitochondria, lysosomes and peroxisomes, remain intact. The Golgi complex, plasma membrane and reticular organelles vesiculate, forming homogeneous-sized vesicles with the same orientation as exists in vivo. Following homogenization the nucleus is totally and exclusively removed by sedimentation forming a nuclear pellet (NP) (500-1000 g for 10 min). The post-nuclear supernatant (PNS) contains the cytosol and all of the other organelles in free suspension, maintaining their in vivo activities. However, these conditions may not always be desirable. For some experiments it may be important to maintain the structure of the Golgi complex or reticular organelles because vesicles derived from these organelles may not retain